Pamela J. Kling

Human Milk as a Potential Enteral Source of Erythropoietin

In addition to its content of traditional nutrients, milk is a rich source of hormones and peptides, which survive digestion in the neonatal gastrointestinal tract secondary to lower proteolytic activity and increased protein permeability. Previous studies have shown accelerated erythropoiesis or elevated serum erythropoietin (Epo) levels in neonatal (suckling) animals after maternal phlebotomy or maternal hypoxia exposure. We sought to determine whether significant quantities of Epo are present in human milk and whether Epo remains intact under physiologic digestion conditions. Immunoreactive Epo concentrations were determined in 409 human milk samples obtained from mothers of term and premature infants. Samples collected between birth and postpartum d 134 were divided into 11 postpartum day groups. Mean milk-borne Epo concentrations were within the normal range for plasma Epo concentrations and rose with postpartum day (F10,398 = 5.82, p < 0.0001). No differences were observed between milk collected from mothers of premature versus term infants. Estimated weekly human milk-borne Epo intakes approximated the lower range of published parenteral therapeutic doses. In simulated digestion at physiologic pH levels of 3.2, 5.8, and 7.4, milk-borne Epo resisted degradation at 1 and 2 h, compared with baseline. Therefore, we conclude that human milk contains considerable amounts of Epo which resist degradation after exposure to gastric juices at physiologic pH levels. These results support continued investigation into the fate and developmental roles of Epo in human milk.

Anemia in children after transplantation: etiology and the effect of immunosuppressive therapy on erythropoiesis

Abstract: Anemia in children after renal transplantation is more common than previously appreciated. Multiple factors appear to play roles in the development of post‐transplant anemia, the most common of which is absolute and/or functional iron deficiency anemia. Most experts recommend that iron limited anemias in transplant patients should be diagnosed using the same criteria as for chronic renal failure patients. Serum erythropoietin (EPO) levels are expected to normalize after a successful renal transplantation with a normal kidney function, yet both EPO deficiency and resistance have been reported. While no large controlled trials comparing the effect of different immunosuppressive agents on erythropoiesis after transplantation have been performed, generalized bone marrow suppression attributable to azathioprine (AZA), mycophenolate mofetil (MMF), tacrolimus, antithymocyte preparations has been reported. Pure red cell aplasia (PRCA) occurs rarely after transplantation and is characterized by the selective suppression of erythroid cells in the bone marrow. PRCA has been reported with the use of AZA, MMF, tacrolimus, angiotensin converting enzyme inhibitors (ACEI), but not with cyclosporine (CSA) use. Post‐transplant hemolytic uremic syndrome has been reported with orthoclone anti T‐cell antibody (OKT3), CSA and tacrolimus therapy. Viral infections including cytomegalovirus, Epstein–Barr virus and human parvovirus B19 have been reported to cause generalized marrow suppression. Management of severe anemia associated with immunosuppressive drugs generally requires lowering the dose, drug substitution or, when possible, discontinuation of the drug. Because this topic has been incompletely studied, our recommendation as to the best immunosuppressive protocol after renal transplantation remains largely dependent on the clinical response of the individual patient.

Iron deprivation increases erythropoietin production in vitro, in normal subjects and patients with malignancy

Although tissue hypoxia is the major stimulus for erythropoietin (EPO) production, serum EPO (sEPO) levels at any given Hb in iron‐deficiency anaemia are relatively higher than in other anaemias. Iron chelators stimulate erythropoiesis in anaemia of chronic disease via unknown mechanisms. A recent study suggested that deferoxamine (DFO) regulates steady‐state EPO RNA. Here we report that altered intracellular iron balance regulates EPO production both in vitro and in two unique clinical trials. In vitro, both iron chelation with DFO and blockade of Tf‐mediated iron uptake with anti‐Tf receptor antibody 42/6, stimulated EPO production in serum‐deprived hepatoma cells. Conversely, iron repletion by haemin, inhibited EPO production in these cells. In clinical studies, sEPO levels rose in adult volunteers treated with DFO coupled to hydroxyethyl starch (HES‐DFO) and in patients with advanced malignancy treated with anti‐Tf receptor antibody 42/6, in a time‐ and dose‐dependent manner. These studies indicate intracellular iron balance regulates EPO production in humans.

Erythrocyte Zinc Protoporphyrin Is Elevated With Prematurity and Fetal Hypoxemia

Objective. To examine the utility of red blood cell (RBC) zinc protoporphyrin/heme ratio (ZnPP/H) as an indicator of fetal iron status, because unfavorable neurodevelopmental outcomes have been associated with poor iron status at birth, as indicated by low serum ferritin, and because few reliable indicators of fetal and early neonatal iron status exist. Methods. Consecutively studied preterm and term fetuses at delivery included the following groups: (1) control nonhypoxic, (2) fetuses with intrauterine growth retardation (IUGR), and (3) fetuses of insulin-treated mothers (FDM). We hypothesized (1) that rapid growth velocity associated with an accelerated erythropoiesis among normal fetuses will lead to reduced iron delivery to a rapidly expanding RBC mass and higher umbilical cord blood RBC ZnPP/H and (2) that fetuses that are exposed to pathologic hypoxemia will experience an additional increase in erythropoiesis and higher cord ZnPP/H. ZnPP/H was determined on saline-washed cord blood erythrocytes by hematofluorometry and was examined for its relationship with clinical factors and cord blood laboratory measurements indicative of tissue oxygenation (plasma erythropoietin [EPO] and reticulocyte count) and iron status (plasma ferritin and erythrocyte indices). Statistical testing included 1-way analysis of variance, 2-way analysis of variance with covariates, simple linear regression, and multiple regression analysis. Results. Among control group subjects, gestational age at birth was inversely correlated with RBC ZnPP/H and reticulocyte count and positively correlated with ferritin and EPO. Relative to control subjects, IUGR and FDM fetuses at specified gestational age groupings had higher ZnPP/H, lower plasma ferritin, and higher plasma EPO. Statistical modeling of the relationship between ZnPP/H and plasma ferritin among all study groups demonstrated significant impacts of gestational age, plasma EPO, maternal hypertension, and maternal smoking. Conclusions. The inverse association of fetal ZnPP/H with gestational age at birth among control subjects is attributable to erythropoietic stimulation likely as a result of increasing growth velocity at the earliest gestational ages. The relatively higher ZnPP/H observed among fetuses in the IUGR and FDM groups likely is attributable to increased erythropoietic activity secondary to pathologic hypoxemia. Decreased placental iron transfer may also have limited iron availability and contributed to elevated ZnPP/H in the IUGR group. These data support the concept that increased erythropoietic activity and/or limited iron transport may place infants of diabetic mothers and infants with growth retardation at risk for developing systemic iron deficiency later in infancy and in early childhood.

Iron status and the treatment of the anemia of prematurity

Many unanswered issues regarding rhEPO therapy in prematurity remain, including which premature infants best respond to rhEPO, what the long-term effect of decreased erythrocyte transfusions is, how nutritional supplementation optimizes the effect of rhEPO, whether or not rhEpo therapy causes iron deficiency later in life, and whether or not it is safe to supplement with parenteral iron. Further study of rhEPO therapy and iron status in prematurity is necessary.

Insulin-like growth factor-I stimulates erythropoiesis when administered enterally

Background: Insulin-like growth factors I and II (IGF-I and IGF-II) are potent growth factors involved in development. IGF-I stimulates proliferation of erythropoietic progenitors and parenteral IGF-I administration stimulates in vivo erythropoiesis in animals. IGF-I and IGF-II are both present in mammalian milks and when milk-borne, are resistant to neonatal gastrointestinal degradation. Whether milk-borne IGF-I or IGF-II regulates neonatal erythropoiesis in not known. We hypothesized that physiological doses of enteral IGFs stimulate erythropoiesis in suckling rats. Methods: Eight day-old Sprague Dawley rats were artificially fed for 4 days with rat milk substitute (RMS) or RMS supplemented with physiological levels of IGF-I or IGF-II. Rats fed IGF-I and IGF-II were compared to control RMS. Blood and marrow were collected; measures of red cell mass, measures of erythropoietic stimulus, and indices of iron status were measured. Results: Rats fed IGF-I had higher hemoglobin (Hb) levels (100 ± 10 g/l), compared to those fed RMS (94 ± 9) or IGF-II (91 ± 6), p < 0.001. After IGF-I supplementation, red blood cell counts (RBC) (p < 0.04) and hematocrits (p < 0.002) were also higher. Plasma erythropoietin (Epo) levels, reticulocytes, plasma iron and erythrocyte iron incorporation were similar. Conclusion: Intact enteral IGF-I reaches distal erythropoietic tissue resulting in greater red cell mass, but not by increasing plasma Epo levels or by altering cellular iron transport.

Pharmacokinetics and pharmacodynamics of erythropoietin during therapy in an infant with renal failure

We treated an infant with anemia and chronic renal failure with recombinant human erythropoietin (300 to 750 U/kg subcutaneously per week) and iron (6 mg/kg enterally) from 1 to 4 months of age. A suboptimal pharmacodynamic response was seen at the lower dose. This may have been due to developmental erythropoietin pharmacokinetic differences, that is, relatively greater neonatal plasma clearance and steady-state volume of distribution compared with those in adults.

Fate of Milk Borne Erythropoietin in Suckling Rats

Mammalian milk is a rich source of hormones and growth factors which survive proteolytic degradation and are absorbed intact in the neonate.1Human milk contains erythropoietin (Epo), the primary endocrine hormone responsible for erythropoiesis.2Recombinant human erythropoietin (rhEpo) may be an effective treatment for the anemia of prematurity but traditional routes of administration, such as intravenous or subcutaneous dosing have thus far provided disappointing results. In animals, milk borne Epo stimulates erythropoiesis.3Additionally, Epo receptors have been found in gastrointestinal tract, wherein vitrostudies show mitogenic and stimulatory effects of Epo on gastrointestinal cells.4,5We hypothesized that milk borne Epo is distributed both to local GI tissues and to the systemic circulation in the Sprague Dawley suckling rat.

Effects of Milk-Borne Physiological Concentrations of Insulin-Like Growth Factors-I or -II (IGF-I, -II) Upon Growth in the Artificially Fed (AR) Suckling Rat

Insulin-Like Growth Factors-I and -II are potent mitogenic peptides that are synthesized by many cell types in mammals (Sara and Hall, 1990, Styne, 1998). These hormones appear to be important in growth regulation of the organism, including during perinatal life (Cohick, et al., 1993,Philipps, et al., 1988). This presumption is strengthened by recent observations of mice bearing nonfunctional genes for IGF-I, -II (Baker, et al., 1993,Lau, et al., 1994), since these have significant growth restriction as well as a high perinatal mortality rate. Attention has also recently focused upon the presence of IGF’s in milk of many species in biologically relevant concentrations. Studies from our laboratories (Philipps, et al., 1991) as well as others (Donovan, et al., 1990) have shown that IGF-I and -II are present in the milk of at least several species. IGF’s are present in most biological fluids in close association with binding proteins and it has been suggested that these binding proteins play a significant role in modulation of IGF-receptor interaction and as a potential reservoir for IGF in the circulation. The serum concentration of IGFBP, particularly BP2 in the suckling, may be responsive to levels of nutritional sufficiency.